Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages

Autoři: Violaine Dubois aff001;  Alexandre Pawlik aff002;  Anouchka Bories aff001;  Vincent Le Moigne aff001;  Odile Sismeiro aff003;  Rachel Legendre aff003;  Hugo Varet aff003;  María del Pilar Rodríguez-Ordóñez aff005;  Jean-Louis Gaillard aff001;  Jean-Yves Coppée aff003;  Roland Brosch aff002;  Jean-Louis Herrmann aff001;  Fabienne Girard-Misguich aff001
Působiště autorů: Université Paris-Saclay, UVSQ, Inserm, Infection et inflammation, Montigny-Le-Bretonneux, France aff001;  Institut Pasteur, Unité de Pathogénomique Mycobactérienne intégrée, UMR3525 CNRS, Paris, France aff002;  Institut Pasteur—Bioinformatics and Biostatistics Hub—C3BI, USR 3756 IP CNRS, Paris, France aff003;  Institut Pasteur—Transcriptome and Epigenome Platform—Biomics Pole—CITECH, Paris, France aff004;  Laboratoire d'Écologie, Systématique et Évolution, Université Paris-Saclay, Orsay, France aff005;  AP-HP. GHU Paris Saclay, Hôpital Ambroise Paré, Boulogne Billancourt, France aff006;  AP-HP. GHU Paris Saclay, Hôpital Raymond Poincaré, Garches, France aff007
Vyšlo v časopise: Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages. PLoS Pathog 15(11): e32767. doi:10.1371/journal.ppat.1008069
Kategorie: Research Article
doi: 10.1371/journal.ppat.1008069


Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival.

Klíčová slova:

Autophagic cell death – Gene expression – Gene regulation – Intracellular pathogens – Mycobacteria – Transcriptome analysis – Amoebas


1. Lavania M, Katoch K, Katoch VM, Gupta AK, Chauhan DS, Sharma R, et al. Detection of viable Mycobacterium leprae in soil samples: Insights into possible sources of transmission of leprosy. Infect Genet Evol. 2008;8: 627–631. doi: 10.1016/j.meegid.2008.05.007 18599381

2. Angenent LT, Kelley ST, St Amand A, Pace NR, Hernandez MT. Molecular identification of potential pathogens in water and air of a hospital therapy pool. Proc Natl Acad Sci U S A. National Academy of Sciences; 2005;102: 4860–5. doi: 10.1073/pnas.0501235102 15769858

3. Ben Salah I, Adékambi T, Drancourt M. Mycobacterium phocaicum in therapy pool water. Int J Hyg Environ Health. 2009;212: 439–444. doi: 10.1016/j.ijheh.2008.10.002 19201259

4. Gomila M, Ramirez A, Gasco J, Lalucat J. Mycobacterium llatzerense sp. nov., a facultatively autotrophic, hydrogen-oxidizing bacterium isolated from haemodialysis water. Int J Syst Evol Microbiol. 2008;58: 2769–2773. doi: 10.1099/ijs.0.65857-0 19060055

5. Guidotti TL, Ragain L, de Haas P, van Soolingen D. Communicating with healthcare providers. J Water Health. 2008;6: s53. doi: 10.2166/wh.2008.032

6. Griffith DE, Aksamit T, Brown-Elliott B a, Catanzaro A, Daley C, Gordin F, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med. 2007;175: 367–416. doi: 10.1164/rccm.200604-571ST 17277290

7. Khan IUH, Selvaraju SB, Yadav JS. Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis. J Clin Microbiol. 2005;43: 4466–72. doi: 10.1128/JCM.43.9.4466-4472.2005 16145093

8. Mignard S, Flandrois J-P. A seven-gene, multilocus, genus-wide approach to the phylogeny of mycobacteria using supertrees. Int J Syst Evol Microbiol. 2008;58: 1432–1441. doi: 10.1099/ijs.0.65658-0 18523191

9. Thomson RM, Carter R, Tolson C, Coulter C, Huygens F, Hargreaves M. Factors associated with the isolation of Nontuberculous mycobacteria (NTM) from a large municipal water system in Brisbane, Australia. BMC Microbiol. 2013;13: 89. doi: 10.1186/1471-2180-13-89 23601969

10. Ripoll F, Pasek S, Schenowitz C, Dossat C, Barbe V, Rottman M, et al. Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus. PLoS One. 2009;4: e5660. doi: 10.1371/journal.pone.0005660 19543527

11. Thomas V, McDonnell G. Relationship between mycobacteria and amoebae: ecological and epidemiological concerns. Lett Appl Microbiol. 2007;45: 349–57. doi: 10.1111/j.1472-765X.2007.02206.x 17897376

12. Falkinham JO. Surrounded by mycobacteria: nontuberculous mycobacteria in the human environment. J Appl Microbiol. 2009;107: 356–67. doi: 10.1111/j.1365-2672.2009.04161.x 19228258

13. Eddyani M, De Jonckheere JF, Durnez L, Suykerbuyk P, Leirs H, Portaels F. Occurrence of free-living amoebae in communities of low and high endemicity for Buruli ulcer in southern Benin. Appl Environ Microbiol. 2008;74: 6547–53. doi: 10.1128/AEM.01066-08 18776024

14. Thomas V, Herrera-rimann K, Blanc DS, Greub G. Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network. Appl Env Microbiol. 2006;72: 2428–2438. doi: 10.1128/AEM.72.4.2428

15. Pagnier I, Raoult D, La Scola B. Isolation and identification of amoeba-resisting bacteria from water in human environment by using an Acanthamoeba polyphaga co-culture procedure. Environ Microbiol. 2008;10: 1135–1144. doi: 10.1111/j.1462-2920.2007.01530.x 18279351

16. Adékambi T, Ben Salah S, Khlif M, Raoult D, Drancourt M. Survival of environmental mycobacteria in Acanthamoeba polyphaga. Appl Environ Microbiol. 2006;72: 5974–5981. doi: 10.1128/AEM.03075-05 16957218

17. Lamrabet O, Mba Medie F, Drancourt M. Acanthamoeba polyphaga-enhanced growth of Mycobacterium smegmatis. PLoS One. 2012;7: e29833. doi: 10.1371/journal.pone.0029833 22253795

18. Thomas V, Loret J-F, Jousset M, Greub G. Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant. Environ Microbiol. 2008;10: 2728–2745. doi: 10.1111/j.1462-2920.2008.01693.x 18637950

19. White CI, Birtles RJ, Wigley P, Jones PH. Mycobacterium avium subspecies paratuberculosis in free-living amoebae isolated from fields not used for grazing. Vet Rec. 2010;166: 401–402. doi: 10.1136/vr.b4797 20348470

20. Gutierrez MC, Brisse S, Brosch R, Fabre M, Omaïs B, Marmiesse M, et al. Ancient Origin and Gene Mosaicism of the Progenitor of Mycobacterium tuberculosis. PLoS Pathog. Public Library of Science; 2005;1: e5. doi: 10.1371/journal.ppat.0010005 16201017

21. Ripoll F, Pasek S, Schenowitz C, Dossat C, Barbe V, Rottman M, et al. Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus. PLoS One. 2009;4: e5660. doi: 10.1371/journal.pone.0005660 19543527

22. Lamrabet O, Merhej V, Pontarotti P, Raoult D, Drancourt M. The genealogic tree of mycobacteria reveals a long-standing sympatric life into free-living protozoa. PLoS One. 2012;7: e34754. doi: 10.1371/journal.pone.0034754 22511965

23. Siddiqui R, Khan NA. Acanthamoeba is an evolutionary ancestor of macrophages: a myth or reality? Exp Parasitol. 2012;130: 95–7. doi: 10.1016/j.exppara.2011.11.005 22143089

24. Barker J, Brown MRW. Trojan Horses of the microbial world: protozoa and the survival of bacterial pathogens in the environment. Microbiology. 1994;140: 1253–1259. doi: 10.1099/00221287-140-6-1253 8081490

25. Greub G, Raoult D. Microorganisms resistant to free-living amoebae. Clin Microbiol Rev. 2004;17: 413–33. doi: 10.1128/CMR.17.2.413-433.2004 15084508

26. Salah IB, Ghigo E, Drancourt M. Free-living amoebae, a training field for macrophage resistance of mycobacteria. Clin Microbiol Infect. 2009;15: 894–905. doi: 10.1111/j.1469-0691.2009.03011.x 19845701

27. Bakala N’Goma JC, Le Moigne V, Soismier N, Laencina L, Le Chevalier F, Roux A-L, et al. Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice. Roy CR, editor. Infect Immun. American Society for Microbiology; 2015;83: 780–791. doi: 10.1128/IAI.02032-14 25486995

28. Cirillo JD, Falkow S, Tompkins LS, Bermudez LE. Interaction of Mycobacterium avium with environmental amoebae enhances virulence. Infect Immun. 1997;65: 3759–67. 9284149

29. Laencina L, Dubois V, Le Moigne V, Viljoen A, Majlessi L, Pritchard J, et al. Identification of genes required for Mycobacterium abscessus growth in vivo with a prominent role of the ESX-4 locus. Proc Natl Acad Sci. 2018;115: E1002–E1011. doi: 10.1073/pnas.1713195115 29343644

30. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15: 550. doi: 10.1186/s13059-014-0550-8 25516281

31. Tatusov RL, Galperin MY, Natale DA, Koonin E V. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000;28: 33–6. doi: 10.1093/nar/28.1.33 10592175

32. Eoh H, Rhee KY. Multifunctional essentiality of succinate metabolism in adaptation to hypoxia in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 2013;110: 6554–9. doi: 10.1073/pnas.1219375110 23576728

33. Jamet S, Quentin Y, Coudray C, Texier P, Laval F, Daffé M, et al. Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis. J Bacteriol. American Society for Microbiology (ASM); 2015;197: 3797–811. doi: 10.1128/JB.00433-15 26416833

34. Mukhopadhyay S, Nair S, Ghosh S. Pathogenesis in tuberculosis: transcriptomic approaches to unraveling virulence mechanisms and finding new drug targets. FEMS Microbiol Rev. 2012;36: 463–485. doi: 10.1111/j.1574-6976.2011.00302.x 22092372

35. Sherman DR, Mdluli K, Hickey MJ, Barry CE, Stover CK. AhpC, oxidative stress and drug resistance in Mycobacterium tuberculosis. Biofactors. 1999;10: 211–7. 10609885

36. Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan JA, Monahan IM, et al. Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages. J Exp Med. 2003;198: 693–704. doi: 10.1084/jem.20030846 12953091

37. Sousa S, Bandeira M, Carvalho PA, Duarte A, Jordao L. Nontuberculous mycobacteria pathogenesis and biofilm assembly. Int J Mycobacteriology. No longer published by Elsevier; 2015;4: 36–43. doi: 10.1016/J.IJMYCO.2014.11.065 26655196

38. Roux A-L, Viljoen A, Bah A, Simeone R, Bernut A, Laencina L, et al. The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages. Open Biol. The Royal Society; 2016;6: 160185. doi: 10.1098/rsob.160185 27906132

39. Dubois V, Viljoen A, Laencina L, Le Moigne V, Bernut A, Dubar F, et al. MmpL8MAB controls Mycobacterium abscessus virulence and production of a previously unknown glycolipid family. Proc Natl Acad Sci U S A. 2018;115: E10147–E10156. doi: 10.1073/pnas.1812984115 30301802

40. Viljoen A, Dubois V, Girard-Misguich F, Blaise M, Herrmann J-L, Kremer L. The diverse family of MmpL transporters in mycobacteria: from regulation to antimicrobial developments. Mol Microbiol. 2017;104. doi: 10.1111/mmi.13873

41. Wei J, Dahl JL, Moulder JW, Roberts EA, O’Gaora P, Young DB, et al. Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages. J Bacteriol. 2000;182: 377–84. doi: 10.1128/jb.182.2.377-384.2000 10629183

42. Wu S, Barnes PF, Samten B, Pang X, Rodrigue S, Ghanny S, et al. Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth. Microbiology. Microbiology Society; 2009;155: 1272–81. doi: 10.1099/mic.0.024638-0 19332828

43. Shin D-M, Jeon B-Y, Lee H-M, Jin HS, Yuk J-M, Song C-H, et al. Mycobacterium tuberculosis Eis Regulates Autophagy, Inflammation, and Cell Death through Redox-dependent Signaling. Deretic V, editor. PLoS Pathog. Public Library of Science; 2010;6: e1001230. doi: 10.1371/journal.ppat.1001230 21187903

44. Chalut C. MmpL transporter-mediated export of cell-wall associated lipids and siderophores in mycobacteria. Tuberculosis (Edinb). 2016;100: 32–45. doi: 10.1016/ 27553408

45. Owens CP, Chim N, Graves AB, Harmston CA, Iniguez A, Contreras H, et al. The mycobacterium tuberculosis secreted protein Rv0203 transfers heme to membrane proteins MmpL3 and MmpL11. J Biol Chem. 2013;288: 21714–21728. doi: 10.1074/jbc.M113.453076 23760277

46. Wright CC, Hsu FF, Arnett E, Dunaj JL, Davidson PM, Pacheco SA, et al. The Mycobacterium tuberculosis MmpL11 Cell Wall Lipid Transporter Is Important for Biofilm Formation, Intracellular Growth, and Nonreplicating Persistence. Ehrt S, editor. Infect Immun. 2017;85: e00131–17. doi: 10.1128/IAI.00131-17 28507063

47. Yamaryo-Botte Y, Rainczuk AK, Lea-Smith DJ, Brammananth R, van der Peet PL, Meikle P, et al. Acetylation of Trehalose Mycolates Is Required for Efficient MmpL-Mediated Membrane Transport in Corynebacterineae. ACS Chem Biol. 2015;10: 734–746. doi: 10.1021/cb5007689 25427102

48. Kumar A, Farhana A, Guidry L, Saini V, Hondalus M, Steyn AJC. Redox homeostasis in mycobacteria: the key to tuberculosis control? Expert Rev Mol Med. Cambridge University Press; 2011;13: e39. doi: 10.1017/S1462399411002079 22172201

49. Geiman DE, Raghunand TR, Agarwal N, Bishai WR. Differential Gene Expression in Response to Exposure to Antimycobacterial Agents and Other Stress Conditions among Seven Mycobacterium tuberculosis whiB-Like Genes. Antimicrob Agents Chemother. 2006;50: 2836–2841. doi: 10.1128/AAC.00295-06 16870781

50. Sherman DR, Voskuil M, Schnappinger D, Liao R, Harrell MI, Schoolnik GK. Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin. Proc Natl Acad Sci U S A. 2001;98: 7534–9. doi: 10.1073/pnas.121172498 11416222

51. Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol. 2003;48: 77–84. doi: 10.1046/j.1365-2958.2003.03425.x 12657046

52. Gouzy A, Poquet Y, Neyrolles O. A central role for aspartate in Mycobacterium tuberculosis physiology and virulence. Front Cell Infect Microbiol. Frontiers Media SA; 2013;3: 68. doi: 10.3389/fcimb.2013.00068 24187657

53. Manca C, Paul S, Barry CE, Freedman VH, Kaplan G, Kaplan G. Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro. Infect Immun. American Society for Microbiology (ASM); 1999;67: 74–9. 9864198

54. Ribet D, Cossart P. Pathogen-Mediated Posttranslational Modifications: A Re-emerging Field. Cell. Cell Press; 2010;143: 694–702. doi: 10.1016/j.cell.2010.11.019 21111231

55. Müller MP, Peters H, Blümer J, Blankenfeldt W, Goody RS, Itzen A. The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b. Science. American Association for the Advancement of Science; 2010;329: 946–9. doi: 10.1126/science.1192276 20651120

56. Parra J, Marcoux J, Poncin I, Canaan S, Herrmann JL, Nigou J, et al. Scrutiny of Mycobacterium tuberculosis 19 kDa antigen proteoforms provides new insights in the lipoglycoprotein biogenesis paradigm. Sci Rep. Nature Publishing Group; 2017;7: 43682. doi: 10.1038/srep43682 28272507

57. Goldberg AL, St. John AC. Intracellular Protein Degradation in Mammalian and Bacterial Cells: Part 2. Annu Rev Biochem. Annual Reviews 4139 El Camino Way, P.O. Box 10139, Palo Alto, CA 94303–0139, USA; 1976;45: 747–804. doi: 10.1146/ 786161

58. Neckers L, Tatu U. Molecular chaperones in pathogen virulence: emerging new targets for therapy. Cell Host Microbe. NIH Public Access; 2008;4: 519–27. doi: 10.1016/j.chom.2008.10.011 19064253

59. Ducati RG, Breda A, Basso LA, Santos DS. Purine Salvage Pathway in Mycobacterium tuberculosis. Curr Med Chem. 2011;18: 1258–75. doi: 10.2174/092986711795029627 21366536

60. Rhee KY, Erdjument-Bromage H, Tempst P, Nathan CF. S-nitroso proteome of Mycobacterium tuberculosis: Enzymes of intermediary metabolism and antioxidant defense. Proc Natl Acad Sci. 2005;102: 467–472. doi: 10.1073/pnas.0406133102 15626759

61. Shah P, Swiatlo E. MicroReview A multifaceted role for polyamines in bacterial pathogens. 2008; doi: 10.1111/j.1365-2958.2008.06126.x

62. Rohmer M, Knani M, Simonin P, Sutter B, Sahm H. Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J. Portland Press Ltd; 1993;295: 517–24. doi: 10.1042/bj2950517 8240251

63. Gershenzon J, Dudareva N. The function of terpene natural products in the natural world. Nat Chem Biol. 2007;3: 408–414. doi: 10.1038/nchembio.2007.5 17576428

64. Berney M, Weimar MR, Heikal A, Cook GM. Regulation of proline metabolism in mycobacteria and its role in carbon metabolism under hypoxia. Mol Microbiol. 2012;84: 664–81. doi: 10.1111/j.1365-2958.2012.08053.x 22507203

65. Rossier O, Cianciotto NP. The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection. Infect Immun. American Society for Microbiology (ASM); 2005;73: 2020–32. doi: 10.1128/IAI.73.4.2020-2032.2005 15784543

66. Le Moigne V, Belon C, Goulard C, Accard G, Bernut A, Pitard B, et al. MgtC as a Host-Induced Factor and Vaccine Candidate against Mycobacterium abscessus Infection. Ehrt S, editor. Infect Immun. 2016;84: 2895–2903. doi: 10.1128/IAI.00359-16 27481243

67. Medjahed H, Singh AK. Genetic manipulation of Mycobacterium abscessus. Curr Protoc Microbiol. 2010;Chapter 10: Unit 10D.2. doi: 10.1002/9780471729259.mc10d02s18 20812214

68. Rowbotham TJ. Isolation of Legionella pneumophila from clinical specimens via amoebae, and the interaction of those and other isolates with amoebae. J Clin Pathol. 1983;36: 978–986. doi: 10.1136/jcp.36.9.978 6350372

69. Dubois V, Laencina L, Bories A, Le Moigne V, Pawlik A, Herrmann J-L, et al. Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes. J Vis Exp. 2018; doi: 10.3791/57766 30320743

70. Benjamini, Hochberg. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J R Stat Soc Ser B. 1995;57: 289–300.

71. Vallenet D, Engelen S, Mornico D, Cruveiller S, Fleury L, Lajus A, et al. MicroScope: a platform for microbial genome annotation and comparative genomics. Database (Oxford). Oxford University Press; 2009;2009: bap021. doi: 10.1093/database/bap021 20157493

72. Alexa A, Rahnenfuhrer J. topGO: Enrichment Analysis for Gene Ontology. R package version 2.36.0. 2019.

73. Simeone R, Sayes F, Song O, Gröschel MI, Brodin P, Brosch R, et al. Cytosolic access of Mycobacterium tuberculosis: critical impact of phagosomal acidification control and demonstration of occurrence in vivo. PLoS Pathog. 2015;11: e1004650. doi: 10.1371/journal.ppat.1004650 25658322

Hygiena a epidemiologie Infekční lékařství Laboratoř

Článek vyšel v časopise

PLOS Pathogens

2019 Číslo 11

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Zvyšte si kvalifikaci online z pohodlí domova

Antiseptika a prevence ve stomatologii
nový kurz
Autoři: MUDr. Ladislav Korábek, CSc., MBA

Citikolin v neuroprotekci a neuroregeneraci: od výzkumu do klinické praxe nejen očních lékařů
Autoři: MUDr. Petr Výborný, CSc., FEBO

Zánětlivá bolest zad a axiální spondylartritida – Diagnostika a referenční strategie
Autoři: MUDr. Monika Gregová, Ph.D., MUDr. Kristýna Bubová

Diagnostika a léčba deprese pro ambulantní praxi
Autoři: MUDr. Jan Hubeňák, Ph.D

Význam nemocničního alert systému v době SARS-CoV-2
Autoři: doc. MUDr. Helena Lahoda Brodská, Ph.D., prim. MUDr. Václava Adámková

Všechny kurzy
Kurzy Doporučená témata Časopisy
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se